Tissue factor inflammatory response regulated by promoter genotype and p38 MAPK in neonatal vs. adult microvascular endothelial cells.

OBJECTIVE AND DESIGN: Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18-basepair deletion (D) or insertion (I) at -1,208. We sought to determine the relationship between these haplotypes and interleukin-1α (IL-1α)-induced TF expression in neonatal versus adult HMVEC. RESULTS: IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed threefold higher levels of TF mRNA, twofold higher TF protein, and fourfold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates, but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. CONCLUSIONS: Upregulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants.

Expression of cytokines by multipotent neural progenitor cells.

Recent work with mammalian neural stem cells has highlighted the role of cytokine signaling in the proliferation and differentiation of these multipotent cells. While the responsiveness of neural progenitors to exogenously applied growth factors has been demonstrated in vivo as well as in vitro, little attention has been given to the production of cytokines by these cells. Here we use immunocytochemistry, RT-PCR, and ELISA to show that under standard growth conditions multipotent neural progenitor cells from humans express multiple cytokines including IL-1alpha, IL-1beta, IL-6, TGF-beta1, TGF-beta2, TNF-alpha, but not IL-2, IL-4, or IFN-gamma. Neural progenitor cells from rat and mouse express some, but not all, of these cytokines under similar conditions. While the function of cytokine expression by neural progenitor cells remains to be elucidated, these signaling molecules are known to be involved in neural development and may play a role in the activation of quiescent stem cells by a variety of pathological processes.

The immunological properties of adult hippocampal progenitor cells.

Adult hippocampal progenitor cells (AHPCs) derived from mature rats were studied in mixed co-cultures and shown not to elicit a proliferative response from human peripheral blood mononuclear cells (PBMCs) or allogeneic spleen cells. FACS analysis revealed low class I and no detectable class II (Ia) MHC expression by these cells. RT-PCR showed that AHPCs express the anti-inflammatory cytokine TGF-beta1. AHPCs did not, however, significantly impede the proliferation of OKT3- or PHA-stimulated PBMCs. Taken together, these results indicate that AHPCs are non-immunogenic in vitro. This is consistent with their pattern of MHC expression and does not require an active immunosuppressive mechanism.

Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro.

To determine if megakaryocytes are targeted by immune thrombocytopenic purpura (ITP) autoantibodies, as are platelets, we have studied the effects of ITP plasma on in vitro megakaryocytopoiesis. Umbilical cord blood mononuclear cells were incubated in the presence of thrombopoietin and 10% plasma from either ITP patients (n = 53) or healthy donors. The yield of megakaryocytic cells, as determined by flow cytometry, was significantly reduced in the presence of ITP plasma containing antiplatelet glycoprotein Ib (GPIb) autoantibodies (P <.001) as compared with both the control and patient plasma with no detectable anti-GPIIb/IIIa or anti-GPIb autoantibodies. Platelet absorption of anti-GPIb autoantibodies in ITP plasmas resulted in double the megakaryocyte production of the same plasmas without absorption, whereas platelet absorption of control plasma had no effect on megakaryocyte yield. Furthermore, 2 human monoclonal autoantibodies isolated from ITP patients, 2E7, specific for human platelet glycoprotein IIb heavy chain, and 5E5, specific for a neoantigen on glycoprotein IIIa expressed on activated platelets, had significant inhibitory effects on in vitro megakaryocytopoiesis (P <.001). Taken together, these data indicate that autoantibodies against either platelet GPIb or platelet GPIIb/IIIa in ITP plasma not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.

Expression of melanoma antigen gene by cells from inflamed joints in juvenile rheumatoid arthritis.

OBJECTIVE: To determine if synovial fluid (SF) cells from inflamed joints of patients with juvenile rheumatoid arthritis (JRA) express melanoma antigen gene (MAGE) RNA and protein. METHODS: The pattern of MAGE-A1 expression was analyzed in inflammatory synovial tissue and peripheral blood mononuclear cells (PBMC) from patients with JRA by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry. RESULTS: MAGE-A1, previously detected only in tumor cells, is strongly expressed in SF cells from patients with JRA. Immunocytochemistry revealed strong staining of SF cells in all of 22 specimens tested. PBMC from patients (7 of 7) also expressed MAGE-A1, but not as strongly as SF cells. Two-color immunofluorescence showed colocalization with CD4 and CD14. Flow cytometry on 3 samples of SF cells confirmed the presence of MAGE-A1 on the cell surface and intracellularly. Five of 5 SF cell samples were positive for MAGE-A1 by RT-PCR. CONCLUSION: Mononuclear cells from inflamed joints and blood from patients with JRA express MAGE-A1. MAGE family proteins were previously thought to be expressed only by certain tumors and presented by HLA Class I, resulting in tumor cell lysis by cytotoxic T cells. The observation of MAGE-A1 expression in JRA suggests an association with autoimmune disease and a possible causal role for MAGE antigen in the chronic inflammation of JRA.